Axio zoom v16 stereo

Leaf samples were stained for iron using Perls' reagent as previously described (Meguro et al., 2007 (link)). Samples were mounted in glycerol (50% v/v) and imaged on an Axio Zoom.V16 stereo microscope with an Axiocam 512 color camera (Zeiss, Cambourne, UK). For measuring iron concentrations, dried leaf samples were digested in 0.25 ml nitric acid (69% w/v) and 0.25 ml hydrogen peroxide (30% w/v) at 90°C. After neutralizing with 1 ml ammonium acetate (15% w/v), samples were reduced with 0.1 ml ascorbic acid (4% w/v). Fe²⁺ was quantified using the colorimetric iron chelator ferene (3‐(2‐pyridyl)‐5,6‐bis‐[2‐(5‐furyl‐sulfonic acid)]‐1,2,4‐triazine, 0.1% w/v) and absorbance was measurement at 593 nm. Iron and other elements in pea seeds were quantified by Inductively Coupled Plasma‐Optical Emission Spectroscopy following digestion of ground samples in nitric acid (55% w/v) and hydrogen peroxide (6% w/w) at 95°C for 16 h.

Six virgin females and four males of each genotype were reared for 2 days on a standard diet at 25°C. The flies were removed, and the vials were kept at 25°C. Shortly before hatching, female pupae were scored and collected on double-sided sticky tape on a glass slide, briefly frozen at −20°C and analyzed with a ZEISS AxioZoom V16 stereo microscope.

Videos at 100–250 frame per second of beating hearts were taken from live 72 hr post-fertilization (hpf) embryos using an Axiozoom V16 Stereo (Carl Zeiss, Oberkochen, Germany) with a 10× objective lens and an Orca Flash 4.0T high-speed digital camera (Hamamatsu Photonics, Hamamatsu City, Shizuoka, Japan). Confocal images were collected on a Leica TCS SP5 with a 63x objective lens. To assess motility, embryos were tapped gently with a whisker.